Project Summary: In small vessel stroke (SVS), which accounts for 20% of ischemic strokes, tissue plasminogen activator (tPA) is ineffective because it can take a prohibitively long time to diffuse to the clot, and catheter-based thrombectomy devices cannot access small vessels. Moreover, treatment associated hemorrhaging limits tPA use to within a few hours of the onset of symptoms for all ischemic strokes. As a result, there is an urgent need for strategies that overcome these limitations, particularly in SVS, while reducing the risks associated with tPA. Building on a successful previous work, a drug delivery strategy is proposed that can selectively target small artery occlusions and deliver mechanical force to accelerate thrombolysis. The objective of this proposal is to investigate and test within realistic models an approach where injected, dispersed magnetic beads are assembled into blood cell sized microwheels (wheels) capable of targeting occlusive clots located in small vessels and lysing them with a combination of mechanical and biochemical action. The central hypothesis is that wheels can (i) target occluded small arteries by exploiting the low flow regions at the entrance of these vessels, (ii) achieve reperfusion at rates an order-of-magnitude faster than soluble tPA, and (iii) improve outcomes in murine models of stroke. This hypothesis will be tested with the following specific aims: Aim 1. Identify magnetic field conditions for wheels targeting of occlusions. Wheels will be assembled in flowing blood and directed to occluded channels or vessels. Microfluidic, zebrafish, and 3D human cerebrovascular models will be used to test the assembly and targeting. Aim 2. Determine rates for thrombolysis of occlusive thrombi using tPA functionalized wheels. It is postulated that tPA functionalized wheels can dissolve fibrin- and platelet-rich clots within microfluidic models and achieve reperfusion in zebrafish and 3D human cerebrovascular models, at rates significantly faster than soluble tPA. Aim 3. Measure the functional benefit of wheel thrombolysis in vivo. In comparison to soluble tPA, wheel mediated thrombolysis will improve safety, motor, and neurological outcomes in murine stroke models and can be visualized using high-resolution MRI and micro-CT. In Aims 1 and 2 the expected outcomes are identifying the operating conditions for wheel assembly, targeting, and fibrinolysis that provide faster reperfusion compared to tPA and can be scaled-up to human-size vascular networks. In Aim 3, it will be shown that wheel thrombolysis is a superior strategy to systemic administration of tPA in terms of neurobehavioral outcomes in a stroke model and can be imaged in vivo. This approach is significant because it could lead to the development of a more rapid and less invasive strategy for alleviating ischemia than methods currently available. This approach is innovative because of the use of external magnetic fields to propel fibrinolytic microdevices to the sites of occlusion and provide mechanical action to accelerate reperfusion time compared to systemic administration of tPA.